Pseudomonas brassicacearum Strain DF41 Kills Caenorhabditis elegansthrough Biofilm-Dependent and Biofilm-Independent Mechanisms

Munmun Nandi,a Chrystal Berry,b Ann Karen C. Brassinga,a Mark F. Belmonte,c W. G. Dilantha Fernando,d Peter C. Loewen,a

Teresa R. de Kievita

Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canadaa; Enteric Diseases Division, National Microbiology Laboratory, Public Health Agency ofCanada, Winnipeg, Manitoba, Canadab; Department of Biological Science, University of Manitoba, Winnipeg, Manitoba, Canadac; Department of Plant Science, Universityof Manitoba, Winnipeg, Manitoba, Canadad

ABSTRACT

Pseudomonas brassicacearum DF41 is a biocontrol agent that suppresses disease caused by the fungal pathogen Sclerotinia scle-rotiorum. A number of exometabolites are produced by DF41, including the lipopeptide sclerosin, hydrogen cyanide (HCN), anddegradative enzymes. The production of these compounds is controlled at both the transcriptional and posttranscriptional levelsby quorum sensing (QS) and the Gac two-component regulatory system. In order to be successful, a biocontrol agent must per-sist in the environment at levels sufficient for pathogen control. Bacterivorous predators, including nematodes, represent a chal-lenge to the establishment of introduced microorganisms. In the current study, DF41 was investigated for its ability to resist pre-dation by Caenorhabditis elegans. We discovered that this bacterium is capable of killing C. elegans through two differentmechanisms: the first involves exposure to toxic metabolites, and the second entails biofilm formation on the nematode headblocking the buccal cavity. Biofilm formation on nematodes, which has been reported only for Yersinia spp. and Xenorhabdusnematophila, is dependent upon the Gac system. Biofilms were not observed when bacteria were grown on NaCl-containing me-dium or on C. elegans biofilm-resistant mutants. Coculturing with nematodes led to the increased expression of the pdfRI-rfiAQS genes and hcnA, which is under QS control. HCN was the most nematicidal of the exometabolites, suggesting that this bacte-rium can respond to predator cues and upregulate expression of toxins accordingly. In summary, DF41 is able to respond to thepresence of C. elegans, and through two distinct mechanisms, it can escape predation.

IMPORTANCE

Pseudomonas brassicacearum DF41 can suppress fungal pathogens through a process known as biocontrol. To be successful, abiocontrol agent must be able to persist in the environment at levels sufficient for pathogen control. Predators, including thenematode Caenorhabditis elegans, represent a threat to persistence. The aim of the current study was to investigate the DF41-C.elegans interaction. We discovered that DF41 is able to escape predation through two distinct mechanisms. The first involvesexposure to toxic bacterial metabolites, and the second entails the formation of a sticky coating on the nematode head, called abiofilm, which blocks feeding and causes starvation. We report here a pseudomonad forming biofilms on the C. elegans surface.When grown with C. elegans, DF41 exhibits altered gene expression and metabolite production, indicating that this bacteriumcan sense the presence of these predators and adjust its physiology accordingly.

The production of extracellular metabolites by biocontrol bac-teria is energetically costly; as such, these compounds are ex-

pected to impart a fitness advantage to the producer. It can beargued that reduced competition for resources is not sufficient towarrant the synthesis of inhibitory compounds. Rather, theseproducts must provide additional advantages, such as reducingthe threat of grazing predators through their repellent and/or cidalactivities (1, 2). Nematodes are among the most abundant animalson the planet, and through their grazing pressure, they are be-lieved to influence microbial community structure (3). Bacteria,in turn, have evolved defensive mechanisms to resist nematodegrazing, including the production of exometabolites that act todeter and/or reduce predator populations. The model organismCaenorhabditis elegans has been widely employed in bacterium-nematode interaction studies because of its genetic tractabilityand the comprehensive array of mutants available (4). For pseu-domonads that exhibit pathogenicity toward C. elegans, killingensues through two distinct mechanisms. On rich media, whichsupport the production of high levels of toxic metabolites, deathoccurs through intoxication, a mechanism known as fast killing

(5–8). Conversely, low-nutrient media result in the production ofsublethal levels of exometabolites, and slow killing occurs over thecourse of several days. In this instance, bacterial colonization ofthe intestinal tract ultimately leads to nematode death (8).

In nature, the bulk of bacterial biomass exists as an adherentcommunity of cells encased in an extrapolymeric matrix, collec-tively known as a biofilm. The ability to form biofilms has manyadvantages, including protection from environmental assaultsthat would threaten planktonic cells (9). Biofilms exhibit elevated

Received 25 July 2016 Accepted 13 September 2016

Accepted manuscript posted online 16 September 2016

Citation Nandi M, Berry C, Brassinga AKC, Belmonte MF, Fernando WGD, LoewenPC, de Kievit TR. 2016. Pseudomonas brassicacearum strain DF41 killsCaenorhabditis elegans through biofilm-dependent and biofilm-independentmechanisms. Appl Environ Microbiol 82:6889 – 6898. doi:10.1128/AEM.02199-16.

Editor: M. A Elliot, McMaster University

Address correspondence to Teresa R. de Kievit, Teresa.Dekievit@ad.umanitoba.ca.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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resistance to biocidal agents, immune components, desiccation,and UV radiation (9, 10). For a select group of bacteria, the abilityto form biofilms allows them to escape predation by grazing nem-atodes. In 2002, Darby and coworkers reported that Yersiniapestis is able to form biofilms on the head and body of C. elegans,blocking feeding and ultimately causing starvation (11). Simi-larly, Xenorhabdus nematophila (12) and some but not all strainsof Yersinia pseudotuberculosis (13) are capable of establishingnematode-associated biofilms. Screening of an array of 26 patho-genic bacteria revealed that C. elegans surface colonization is not acommon trait (14, 15). Even the notorious infection-related bio-film formers Pseudomonas aeruginosa and uropathogenic Esche-richia coli were found to lack this ability, leading to the conclusionthat this defense strategy is employed by only a few bacterial spe-cies (15).

Pseudomonas brassicacearum strain DF41 is a biocontrol agentcapable of suppressing disease caused by the plant-pathogenicfungus Sclerotinia sclerotiorum (Lib.) de Bary in both greenhouseand field studies (16, 17). This bacterium produces an arsenal ofextracellular metabolites, including degradative enzymes, hydro-gen cyanide (HCN), and a novel lipopeptide called sclerosin (17,18). Sclerosin has been confirmed to be essential for biocontrol, asa sclerosin-deficient mutant, DF41-1278, exhibits dramatically re-duced fungal suppression (17). A complex regulatory cascadeoversees the production of DF41 exometabolites, including theGacS/GacA two-component system, the stationary-phase sigmafactor RpoS, and the stringent response (17, 19). In addition,DF41 has a quorum-sensing (QS) system composed of a LuxR-type transcriptional activator (PdfR) and an acyl-homoserine lac-tone (AHL) synthase (PdfI), encoded by pdfR and pdfI, respec-tively (20). Immediately downstream of pdfI lies a third gene in theQS locus, called rfiA. RfiA belongs to a distinct group of LuxRregulators characterized as having a C-terminal helix-turn-helixDNA binding motif but lacking an N-terminal AHL binding do-main (20, 21). AHL signaling molecules are not involved in DF41biocontrol, because an AHL-deficient strain exhibits no discern-ible phenotype (20). RfiA, on the other hand, was found to beessential, as an rfiA mutation results in a complete loss of antifun-gal activity (20). RfiA is believed to control expression of thePdfABC efflux pump involved in exometabolite export. In the�rfiA mutant background, metabolites that are normally excretedaccumulate to elevated levels within the mutant cells (20).

The aim of the current study was to elucidate the interactionbetween DF41 and the bacterivorous predator C. elegans. Specifi-cally, we were interested to learn whether DF41 is able to resistnematode grazing and what role, if any, DF41 exoproducts play inthe bacterium-nematode interaction. We discovered that DF41 iscapable of killing C. elegans through two distinct modes; the firstinvolves exposure to toxic metabolites, while the second entailsbiofilm formation on the nematode surface. It has been reportedthat bacteria are capable of responding to predator cues throughaltered expression of toxin-encoding genes (7, 22, 23). To see if thesame holds true for DF41, we cocultured bacteria with C. elegansand monitored the expression of genes associated with exome-tabolite production. In the presence of C. elegans, several geneswere upregulated, including hcnA (hydrogen cyanide) and the QSgenes pdfRI and rfiA. Our findings indicate that through solublechemical cues and/or direct contact, DF41 is able to perceive thepresence of C. elegans and adjust its physiology accordingly.

MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains and plas-mids used in this study are listed in Table 1. E. coli was cultured at 37°C onlysogeny broth (LB) agar (Difco Laboratories, Detroit, MI). Pseudomonasstrains were routinely cultured on LB, King’s B (KB) (24), or in M9 min-imal salts medium supplemented with 0.2% glucose (M9-Glc) at 28°C,unless otherwise indicated. The antibiotics used were 100 �g/ml ampicil-lin (Amp), 15 �g/ml gentamicin (Gm), and 15 �g/ml tetracycline (Tc) forE. coli, and 20 �g/ml Gm, 40 �g/ml piperacillin (Pip), and 15 �g/ml Tc forDF41. All antibiotics were obtained from Research Products InternationalCorp. (Mt. Prospect, IL).

Nematode strains and culture conditions. The C. elegans strains usedin this work include wild-type Bristol N2, AT6 [srf-2(yj262)], DC9 [bah-3(br9)], and CB4856, which were maintained at 15°C on nematodegrowth medium (NGM) (25) supplemented with E. coli OP50. The pro-tocols available in WormBook were used in order to obtain the synchro-nous cultures (26). L4-stage hermaphrodites were used in the studies de-scribed herein.

Nucleic acid manipulation. Cloning, purification, electrophoresis,and other manipulations of DNA were performed using standard tech-niques (27).

Construction of a DF41 hcn mutant. To generate an hcn mutant, aninternal region of the DF41 hcn gene cluster was PCR amplified using prim-ers hcnA-FOR1 and hcnC-REV1 (Table 1). A TOPO kit (Thermo FisherScientific, Waltham, MA, USA) was employed to clone the 1.6-kb PCRproduct into the pCR2.1-TOPO vector, creating pCRhcnABC-41.To liberate the insert, pCRhcnABC-41 was digested with HindIIIand XhoI and subcloned into the same sites of the suicide vectorpKNOCK-Tc. Triparental mating between the donor [E. coli DH5��pir(pKNOCKhcnABC)], helper [E. coli DH5�(pRK600)], and recipi-ent (DF41) strains was performed to interrupt the wild-type hcnABC genecluster as a single crossover insertion selected using Tc. PCR was used toconfirm the hcn mutation, and the mutant was found to be devoid of HCNproduction when tested using Cyantesmo paper (Macherey-Nagel GmbHand Co., Germany).

Nematode slow- and fast-killing assays. Slow-killing assays were per-formed by spotting 10 �l of a 1:10 dilution of an overnight bacterialculture grown in KB, LB, or NGM onto a 35 by 10-mm agar plate of thesame medium. After 24 h of incubation at 28°C, the plates were cooled toroom temperature and seeded with 30 L4-stage nematodes. The plateswere incubated at 20°C, and the nematodes were scored for viability byexamining them with a stereomicroscope over a 10-day period. Nema-todes without detectable movement were considered dead after confirma-tory prodding with a nematode pick. Three replicates were included foreach trial, and the assays were repeated three times independently. Fast-killing assays were carried out in a similar manner, except that brain heartinfusion (BHI) agar (Difco) with and without FeCl3 (100 �M) was used asthe medium, and nematodes were monitored at 4-, 6-, 8-, 10-, 12-, and24-h time points.

Microscopic imaging of Caenorhabditis elegans. Bacterial strainsharboring pMCh-23, which encodes constitutively expressed mCherryfluorescent protein, were spotted onto agar plates and incubated over-night at 28°C. Plates were cooled to room temperature prior to seedingwith nematodes, followed by incubation at 20°C. Nematodes weremounted onto 2% agarose pads on glass microscope slides and anesthe-tized with 10 mmol/liter levamisole (Sigma) in M9 medium. The nema-todes were examined with a Zeiss LSM 700 confocal laser scanning micro-scope (Carl Zeiss Microscopy GmbH, Göttingen, Germany).

Biofilm formation. To analyze the ability of DF41 and derivativestrains to form biofilms, a 96-well plate assay was employed, as detailed ina study by Berry et al. (17).

Analysis of metabolite and regulatory gene expression in DF41 inthe presence and absence of Caenorhabditis elegans. Gene expressionwas monitored in DF41 grown in the presence and absence of C. elegans.Approximately 200 nematodes were added to a 3-ml volume of DF41

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culture (optical density at 600 nm [OD600], 0.1; M9-Glc), which was in-cubated at room temperature for 72 h with shaking. A 1-ml volume ofculture was spun for 1 min at 1,000 � g to pellet the nematodes, afterwhich 400 �l of the bacterial cell suspension was added to 800 �l ofRNAprotect (Qiagen, Valencia, CA, USA). Total RNA was extracted usingthe RNeasy minikit (Qiagen), and residual genomic DNA was removed bytreatment with Turbo RNase-free DNase I (Ambion, Carlsbad, CA, USA).

cDNA was generated by reverse transcription using the Maxima first-strand cDNA synthesis kit (Thermo Scientific, Rockford, IL, USA) underthe following conditions: initial heating at 25°C for 10 min, 50°C for 15min for reverse transcription, and 85°C for 5 min for enzyme denatur-ation. The primer sequences for the genes of interest are listed in Table 1.Reverse transcription-quantitative PCR (qRT-PCR) was performed usinga CFX Connect real-time system (Bio-Rad, Ontario, Canada) and SsoFast

TABLE 1 Bacterial strains, plasmids, and primers used in the study

Strain, plasmid, or primer Relevant genotype, phenotype, or sequencea Reference or source

StrainsC. elegans

N2 Wild-type isolate CGC, University of Minnesota, MNAT6 srf-2(yj262) CGC, University of Minnesota, MNDC9 bah-3(br9) CGC, University of Minnesota, MNCB4856 Wild-type Hawaiian isolate CGC, University of Minnesota, MN

P. brassicacearumDF41 Rifr wild type (canola root tip isolate) 16DF41-1278 Rifr lp::Tn5-1063 genomic insertion 17DF41hcn DF41 with the pKNOCK-Tc vector inserted into the hcn gene This studyAI-deficient mutant DF41 carrying pME6863 20DF41rfiA DF41 with Gmr cassette inserted into rfiA gene 20DF41gacS Rifr gacS::Tn5-1063 genomic insertion 17DF41-rfp DF41 containing mCherry expressed from pMCh-23 18DF41-1278-rfp DF41-1278 containing mCherry expressed from pMCh-23 18DF41hcn-rfp DF41hcn containing mCherry expressed from pMCh-23 This studyAI-deficient-rfp mutant DF41-6863 containing mCherry expressed from pMCh-23 This studyDF41rfiA-rfp DF41rfiA containing mCherry expressed from pMCh-23 This studyDF41gacS-rfp PA23gacS containing mCherry expressed from pMCh-23 This study

E. coliDH5� supE44 �U169 (�80lacZ�M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 GibcoDH5� �pir �pir lysogen of DH5� 46OP50 Laboratory strain for maintenance of C. elegans; uracil auxotroph 25

Chromobacterium violaceum CVO26 Autoinducer synthase (cviI) mutant from C. violaceum ATCC 31532autoinducer biosensor

29

PlasmidspME6863 pME6000 carrying the aiiA gene from Bacillus sp. A24 under the

constitutive Plac promoter47

pCR2.1 TA cloning vector, Ampr InvitrogenpKNOCK-Tc Suicide vector for insertional mutagenesis; R6K ori Rp4 oriT Tcr 48pRK600 Contains tra genes for mobilization, Chlr 49pCRhcnABC 1.6-kb hcnABC fragment in pCR2.1 This studypKNOCK-hcnABC 1.6-kb fragment from hcnABC in pKNOCK-Tc This studypUCP23 Broad-host-range vector, Ampr Gmr 50pRSET-B mCherry mCherry expression vector, f1 ori, Ampr 51pMCh-23 pUCP23 carrying the mCherry red fluorescent protein gene 18

PrimershcnA-FOR 5=-ATGGGCGTATGCCACTGC-3= This studyhcnC-REV 5=-TAAGCACACGACGCGCCG-3= This studysclerosin-FOR 5=-CCACAAACGGCATTTGCTGG-3= This studysclerosin-REV 5=-AGTTTGCTAAGGACCGCTGC-3= This studyhcnC-FOR 5=-TACGTGGCGCAGAAAGACAACG-3= This studyhcnC-REV 5=-TTGCACCAACCCTTCGATCTCG-3= This studypdfR-FOR 5=-AGCATCATCGCCAACCAACACC-3= This studypdfR-REV 5=-GTTTTTTCCCAGTGCGAGCCAG-3= This studypdfI-FOR 5=-ACCGTTGACAGACGCAATATCG-3= This studypdfI-REV 5=-AGCGTTCTTGCTAAGGACCTCC-3= This studyrfiA-FOR 5=-GCACCTGAACTTGCCGAACAAC-3= This studyrfiA-REV 5=-GCATCCATCGGATAAGCGAACG-3= This studygacS-FOR 5=-TGGTGCAAACCCTGCTCGAAG-3= This studygacS-REV 5=-TCTGCACGTCCATCAACACCAG-3= This study

a Rifr, rifampin resistance; Tc, tetracycline; Gmr, gentamicin resistance; Ampr, ampicillin resistance; Chlr, chloramphenicol resistance; Carb, carbenicillin.

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EvaGreen Supermix (Bio-Rad). The PCR conditions included an initialdenaturation at 98°C for 2 min, followed by 40 cycles of 98°C for 30 s, 60°Cfor 30 s, and 72°C for 30 s. Reactions were performed in triplicate, andexperiments were repeated three times with four biological replicates.Relative gene expression was determined using the comparative thresholdcycle (CT) method, as described by Livak and Schmittgen (28).

Analysis of HCN and AHL signal production. DF41 was grown in thepresence and absence of C. elegans for 72 h at room temperature on M9-Glc agar plates sealed with Parafilm. HCN production was monitoredusing Cyantesmo paper, which turns blue in the presence of this volatilecompound. The production of AHL molecules was assessed qualitativelyby spotting 5 �l of culture grown for 72 h in the presence and absence ofC. elegans onto plates seeded with Chromobacterium violaceum CVO26.This bacterial strain is able to produce the purple pigment violacein onlyin the presence of exogenous autoinducer (29). Samples were analyzed intriplicate, and the experiments were repeated twice.

Statistical analysis. An unpaired Student t test was used for statisticalanalysis of biofilm formation, qRT-PCR gene expression, and AHL pro-duction. The log rank (Mantel-Cox) test was applied for statistical analysisof pairwise comparisons in the fast and slow nematode killing assays.Nematodes that died from crawling off the plate were excluded fromstatistical analysis.

RESULTSPseudomonas brassicacearum DF41 kills Caenorhabditiselegans through production of toxic metabolites. When DF41and derivative strains, including DF41-1278, DF41hcn, autoin-ducer (AI)-deficient DF41, DF41rfiA, and DF41gacS, were grownon BHI, which supports fast killing by pseudomonads (7, 8), dif-

ferences in lethality were observed; however, none elicited 100%nematode killing at 24 h (Fig. 1A). The lowest level of toxicity wasexhibited by the gacS and hcn mutants. The sclerosin-deficientstrain, DF41-1278, was similar to wild type, indicating that thislipopeptide (LP) does not contribute to fast killing. Similarly, theautoinducer (AI)-deficient DF41 strain (pME6863) exhibited nochange in lethality. The most toxic strain was the �rfiA mutant.HCN production was increased in this background compared tothe DF41 wild type (data not shown), likely accounting for thehigh nematicidal activity.

Although DF41 produces HCN, it does so at levels lower thanwhat we have observed for other biocontrol strains, includingPseudomonas chlororaphis PA23 and Pseudomonas protegens Pf5(data not shown). Supplementing media with iron has been re-ported to elevate HCN production (30); therefore, BHI contain-ing 100 �m FeCl3 was employed in the aforementioned assays tosee if more rapid killing would ensue. While the same relativetoxicity pattern was observed, the presence of iron did enhanceoverall lethality (Fig. 1B). When nematodes were placed on BHI(100 �M FeCl3), the DF41 wild-type and sclerosin mutant strainscaused 100% killing after 12 h. As before, the �rfiA mutant strainwas the most toxic, killing 100% of the nematodes by 10 h, whilethe HCN-deficient hcn and gacS mutants were completely benign(Fig. 1B). Collectively, these findings indicate that on BHI (100�M FeCl3), HCN is the primary compound responsible for fastkilling of C. elegans. Due to reduced production of this volatile

FIG 1 Fast-killing (A and B) and slow-killing (C and D) of Caenorhabditis elegans by Pseudomonas brassicacearum DF41. Kaplan-Meier survival plots of C.elegans N2 (n � 25) fed E. coli OP50, DF41, or derivative strains propagated on BHI agar (A), BHI agar supplemented with 100 �M FeCl3 (B), NGM agar(C), and KB agar (D). Each data point represents the average of three biological replicates; error bars indicate the standard deviation (SD). Experimentswere performed three times; one representative data set is shown. Asterisks indicate significant difference from the wild type as determined by the log ranktest (*, P 0.01; **, P 0.001; ***, P 0.0001). Note that in panels A and B, symbols representing the OP50, �hcn mutant, and �gacS mutant strainsoverlap.

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compound on media containing low FeCl3, however, rapid intox-ication does not occur.

Slow killing of Caenorhabditis elegans by biofilm-dependentand biofilm-independent mechanisms. For the slow-killing as-says, DF41 and mutant strains were grown on NGM and KB agarprior to seeding with nematodes. On NGM agar, the survival ofthe nematodes decreased over the course of days (Fig. 1C); sur-vival rates were strain dependent but nevertheless consistent withlethality mediated by bacterial colonization rather than intoxica-tion. Under these conditions, the gacS mutant was found to be lesstoxic than the hcn mutant. In addition to HCN, the gacS mutantfails to produce degradative enzymes and antibiotic compounds,including sclerosin (17), some of which may be contributing totoxicity. The sclerosin-deficient mutant (DF41-1278) showed re-duced nematicidal effects compared to the wild type, indicatingthat this LP is involved in slow killing, albeit modestly. As before,the greatest toxicity was observed for the �rfiA mutant, whichkilled 100% of the nematodes within 96 h (Fig. 1C).

Slow killing of C. elegans was also observed when nematodeswere fed bacteria growing on KB medium (Fig. 1D). However,there was one dramatic difference compared to nematodes fedDF41 and derivative strains grown on NGM. After 24 h, we no-ticed that biofilms began to form on the C. elegans head and body,which dramatically impacted nematode physiology and behavior(Fig. 2). Microscopic visualization at low magnification revealedthat when nematodes were placed on NGM supplemented withthe standard lab strain E. coli OP50, the nematodes moved freelythrough the bacterial lawns, generating sinusoidal track marksknown as “skd” marks (Fig. 2A). Conversely, on DF41 lawnsgrown on KB medium, aberrant skd marks were formed (Fig. 2Band C), and the nematodes fishtailed through the viscous bacteriallawn like a car moving through deep snow. Moreover, the animalswere frequently observed to migrate away from the bacteria, pos-sibly in an attempt to remove their matrix entrapment (Fig. 2C).Over time, the biofilm-coated nematodes became progressivelythinner, with some disintegrating completely, presumably due tostarvation or exposure to toxic bacterial metabolites (data notshown).

DF41 biofilm formation on Caenorhabditis elegans is depen-dent on GacS. Next, we examined whether all of the DF41 deriv-ative strains were capable of forming biofilms on the C. eleganshead and body. On KB medium, the �hcn, �rfiA, sclerosin-lacking(DF41-1278), and AI-deficient mutant strains all coated the sur-face of C. elegans and resulted in aberrant skd marks (data notshown); however, C. elegans growth on and movement throughthe gacS bacterial lawn resembled those observed for E. coli OP50

(Fig. 2D). Confocal microscopic analysis of red fluorescent pro-tein (RFP)-expressing bacteria revealed that, with the exception ofthe gacS mutant, all of the strains formed a thick biofilm on the C.elegans surface after 24 h (Fig. 3A). In all cases, bacteria are visiblein the mouth, grinder, and intestinal track of the nematodes, in-dicating that bacterial ingestion had occurred prior to biofilm for-mation blocking the buccal cavity.

C. elegans mutants resistant to biofilm formation by Y. pestis, Y.pseudotuberculosis, and X. nematophila have been identified (13,31, 32). To determine whether similar mechanisms of DF41 nem-atode attachment were at play, we examined several biofilm-resis-tant C. elegans strains, namely, the srf-2(yj262) and bah-3(br9)mutants and a naturally biofilm-resistant Hawaiian isolate(CB4856), growing on DF41. In all cases, biofilm formation wasmarkedly reduced on the nematodes (Fig. 3B).

NaCl impacts DF41 biofilm development on the surface ofCaenorhabditis elegans. The fact that biofilms developed on thenematode surface when bacteria were grown on KB, but not BHI,NGM, or LB, suggested that medium composition has an impacton this trait. One obvious difference in the composition of thesemedia is salt content. The three media that did not promote bio-film formation contained NaCl (NGM [0.3%], BHI [0.5%], andLB [1%]), whereas KB medium does not. This finding promptedus to investigate whether NaCl affects the ability of DF41 to formbiofilms on the surface of C. elegans. Nematodes fed DF41 grownon KB medium containing 0.5% NaCl resulted in a noticeabledecrease in biofilm formation on the head and body (Fig. 4A). Theeffects were even more dramatic when the concentration of NaClincreased to 1%, in which case virtually no biofilms were observedon the nematodes (Fig. 4A).

Biofilm formation on abiotic surfaces is affected by NaCl butnot GacS. Factors affecting attachment to abiotic surfaces are ex-pected to be dramatically different from those involved in biofilmformation on biotic surfaces. As such, we employed a 96-well mi-crotiter plate assay to assess the ability of DF41 and derivativestrains to form adherent biomass under different medium condi-tions. While there was no significant difference in the ability of thestrains to form biofilms on plastic, we did notice that in KB, bio-film formation was nearly double that observed for bacteria grownin NGM and LB (data not shown). These findings suggest thatNaCl may be impacting biofilm formation not only on biotic sur-faces but abiotic substrates as well. To test this hypothesis, weexamined the adherent DF41 biomass after 24 h of static growth inKB supplemented with NaCl (0% to 1%). As illustrated in Fig. 4B,a significant decrease in biofilm formation was observed in KBcontaining 1% NaCl. No differences in growth rate were observed

FIG 2 Track marks of nematodes fed on E. coli OP50 (A), DF41 (B and C), and a DF41 gacS mutant (D).

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when bacteria were grown in the presence of NaCl (data notshown); as such, reduced biofilm formation is not a consequenceof altered growth rate.

DF41 gene expression is affected by growth in the presence ofCaenorhabditis elegans. To investigate whether DF41 is able tosense the presence of C. elegans, either through direct contact orsoluble chemical cues, we monitored bacterial gene expressionupon growth in the presence and absence of these predators. Bothregulatory genes (gacS, pdfR, pdfI, and rfiA) and the biosyntheticloci encoding HCN and sclerosin were analyzed. The most dra-

matic increase in gene expression was observed for hcnA, whichwas elevated over 5-fold in the presence of the nematodes;conversely, sclerosin biosynthetic gene activity was not affected(Fig. 5). Expression levels of the QS regulatory genes pdfR, pdfI,and rfiA were all significantly elevated upon C. elegans coculture(Fig. 5). Transcription of gacS, on the other hand, remainedunaffected. We observed increased HCN and AHL signal pro-duction in DF41 cells cocultured with C. elegans (Fig. 6), con-sistent with the increase in hcnA and pdfI transcription, respec-tively.

FIG 3 Microscopic analysis of N2 (A) and �srf-2 mutant (strain AT6) (B) worms fed rfp-tagged DF41 and derivative strains on KB. Confocal images were takenwith Zeiss LSM 700 confocal laser scanning microscope under �10 magnification. Scale bar represents 20 �m.

FIG 4 (A) Microscopic analysis of N2 worms fed rfp-tagged DF41 propagated on KB supplemented with NaCl (0 to 1%). Confocal images were taken with ZeissLSM 700 confocal laser scanning microscope under �10 magnification. Scale bar represents 20 �m. (B) In vitro biofilm formation by DF41 in KB brothsupplemented with NaCl (0 to 1%). Each value represents the mean from three biological replicates standard error. The data point marked with an asteriskindicates statistical significance (P 0.01).

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DISCUSSION

In the current study, we explored the interaction between the bio-control agent P. brassicacearum DF41 and the bacterivorous pred-ator C. elegans. We discovered that DF41 is among a select groupof bacteria capable of establishing biofilms on the C. elegans sur-face, blocking feeding and ultimately starving the nematodes. Tothe best of our knowledge, biofilm formation on C. elegans hasnever been reported for a pseudomonad, pathogenic or otherwise.

It is well established that medium composition has a profoundimpact not only on secondary metabolites produced by pseu-domonads (33) but also the nematicidal activity associated withthese organisms (5–8, 34). Consequently, several different mediawere employed as part of our DF41-C. elegans interaction studies.On nutrient-rich BHI, which has been previously reported to sup-port high exometabolite production and fast killing of C. elegans(6, 7), none of the strains tested led to 100% killing at 24 h (Fig.1A). Because HCN is a nematicidal agent causing rapid paralysisof C. elegans (6, 8), we hypothesized that HCN levels were belowthose required for fast killing. When FeCl3 was added to the mediato boost HCN production, the rate of killing by DF41 was en-hanced such that all of the nematodes were dead by 12 h (Fig. 1B).With respect to relative toxicity, both in the presence and absenceof FeCl3, the strain exhibiting the highest level of nematicidal ac-tivity was the rfiA mutant (Fig. 1A and B). The rfiA mutant exhibitsincreased accumulation of AI molecules and elevated expressionof QS-controlled genes, including pdfI and hcnA (20). HCN anal-ysis revealed elevated production of this volatile by the rfiA mutant(data not shown), which likely accounts for the increased nemati-cidal activity. We cannot rule out the possibility that yet-to-beidentified metabolites trapped within these cells contribute to tox-icity. HCN is clearly an important toxin, since the �hcn and �gacSmutant cells, which are both devoid of HCN production (17),show significantly reduced toxicity, consistent with previous find-ings (6, 8). Sclerosin, on the other hand, did not contribute tokilling under these conditions.

In slow-killing assays, which depend upon infection of the C.elegans intestine, killing occurred over the course of days (Fig. 1Cand D). On both NGM and KB media, HCN, and to a lesser de-gree, sclerosin, contributed to the nematicidal effects; however,one dramatic difference was observed between the two media.After 24 h on KB, biofilms began to accumulate on the surface ofthe nematodes as they translocated through the bacterial lawns.The deleterious effect of biofilm formation is reflected by the factthat with the exception of the gacS mutant and OP50, which donot form biofilms, C. elegans survival decreased on bacteria prop-agated on KB (Fig. 1D).

In Y. pestis, Y. pseudotuberculosis, and X. nematophila, biofilmdevelopment on C. elegans is dependent upon the hmsHFRSgene cluster (11, 32). Analysis of the DF41 genome revealedthe presence of a homologous hmsHFRS operon (hmsH,CD58_RS00620; hmsF, CD58_RS00625; hmsR, CD58_RS00630;and hmsS, CD58_RS00635) (35). This cluster showed the highestdegree of similarity at the amino acid level with PgaABCD of Pseu-domonas fluorescens. The DF41 HmsH protein is 91% identical toPgaA, a predicted poly-beta-1,6-N-acetyl-D-glucosamine (PGA)export porin (accession no. ALI07281.1). HmsF exhibits 94% se-quence identity with PgaB, a PGA N-deacetylase (accession no.WP_024618876.1). HmsR is 99% identical to the PGA synthasePgaC (accession no. ALI07279.1), and HmsS exhibits 93% iden-tity with the PGA biosynthesis protein PgaD (WP_003186997.1).In E. coli, PGA biosynthesis mediated by pgaABCD is essential forbiofilm formation (36, 37). BLAST analysis revealed that ho-mologs of the hmsHFRS operon are found in only a limited num-ber of Pseudomonas species (Table 2). It is noteworthy that thesegenes are absent in P. aeruginosa, which does not form biofilms onC. elegans (15). Conversely, biocontrol strain P. chlororaphisPA23, which harbors this locus, was unable to form biofilms onthe nematode surface under the conditions tested (data not

FIG 5 The impact of C. elegans coculture on DF41 biocontrol gene expression.qRT-PCR analysis was used to quantitatively assess gene expression in thepresence and absence of nematodes. Expression levels in the absence of C.elegans were normalized to 1; differentially expressed genes are indicated withasterisks (*, P 0.05; **, P 0.001). Data shown are the means; error barsindicate SD.

FIG 6 (A) Autoinducer production by DF41 grown in the presence and ab-sence of nematodes detected by the AHL biosensor Chromobacterium viola-ceum CVO26. The mean (SD) of zones of activity (in millimeters) obtainedfrom 10 replicates are shown in the table. The asterisk indicates a significantdifference from the wild type (P 0.001). (B) HCN production by DF41grown in the presence and absence of C. elegans.

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shown). Additional work is required to establish a role for thesegenes in DF41 biofilm development; however, the fact that C.elegans mutants that are resistant to Yersinia and Xenorhabdusbiofilms are resistant to those of DF41 suggests that a commonligand-receptor interaction is involved (15, 32).

In E. coli, a csrA (rsmA) mutant was found to overproduce PGAand showed increased biofilm development (38, 39). CsrA belongsto the RsmA family of repressor proteins that bind mRNA at theribosome-binding site, thereby blocking translation (40). The Gactwo-component system is a positive activator of small RNAs thattitrate out the RsmA proteins allowing target gene expression.Consequently, in a gac mutant, targets remain permanently re-pressed. The findings presented herein suggest that genes under-lying DF41 biofilm formation on the surface of C. elegans are sub-ject to Gac regulation, consistent with the CsrA control observedin E. coli. In another study, the LysR-type regulator NhaR wasfound to activate pgaABCD expression in response to 100 mMNaCl and high pH in E. coli (41). We, on the other hand, observedthat the addition of sodium ions (0.5% [86 mM] and 1% [171mM]) diminished the establishment of DF41 biofilms on bothbiotic and abiotic surfaces (Fig. 4). A negative correlation betweenNaCl and biofilm formation has been reported for Clostridiumdifficile and Streptococcus suis (42, 43). Collectively, these findingssuggest that the impact of sodium on biofilm development is spe-cies dependent.

Prokaryotes and eukaryotes have cohabited the Earth for mil-lions of years; it is not surprising, therefore, that chemical com-munication between the two facilitates mutual perception (44).The production of toxic secondary metabolites is energeticallycostly, and so limiting production to situations where these com-pounds provide a fitness advantage, when confronted by preda-tors, for example, would be beneficial to the producer. We cocul-tured DF41 in the presence of C. elegans to see whether chemicalcues and/or direct contact would lead to changes in the expressionof select bacterial genes. Upon coculture, the most highly upregu-lated DF41 gene was hcnA (Fig. 5). Intriguingly, HCN also exhib-ited the greatest nematicidal activity (Fig. 1). Sclerosin, which ex-hibited almost no toxicity toward C. elegans, was unchanged withrespect to gene expression in DF41 grown with the nematodes.These findings are similar to those reported by Jousset and co-workers, wherein cell-free supernatants of the amoeba Acantham-oeba castellanii were found to increase the expression of phlA (di-acetylphloroglucinol [DAPG]), prnA (pyrrolnitrin), and hcnA inP. fluorescens CHA0 (22). Notably, there was a correlation be-tween the level of expression and the toxicity of the encoded prod-

uct, with the greatest increase in gene expression observed forDAPG, the most toxic metabolite tested (22). More recently,global transcriptomic analysis of P. fluorescens SS101 coculturedwith the protist Naegleria americana revealed altered expression of2.3% of the genome (2). Moreover, protozoan predation led toupregulated lipopeptide and putrescine biosynthesis, with the pu-trescine biosynthesis inducing trophozoite encystment and de-creasing cyst viability (2). When we examined the impact of cocul-turing on DF41 regulatory gene expression, we discovered thatpdfR, pdfI, and the cotranscribed rfiA were upregulated in thepresence of C. elegans (Fig. 5). We have previously shown that hcnexpression is under the control of the PdfRI QS system, whereasthe sclerosin biosynthetic genes are not (20). At present, it is un-clear whether upregulation of the hcn genes is mediated directly orindirectly through increased QS gene expression. Analysis of HCNand AHL signal production by DF41 revealed that both end prod-ucts were upregulated by the presence of the nematodes, consis-tent with our gene expression analysis.

Being encased in an extrapolymeric matrix as a biofilm pro-motes bacterial survival and colonization of root surfaces (45). Inaddition to facilitating exchange of metabolic by-products andgenetic information, this mode of growth affords inhabitants pro-tection from toxic compounds and desiccation. We have made theexciting discovery that DF41 is capable of biofilm formation onthe C. elegans anterior, blocking feeding and ultimately starvingthe animals. Thus, for DF41, biofilm formation offers protectionfrom grazing predators. It is possible that biocontrol agents thatshow increased environmental persistence through biofilm for-mation do so through similar antipredator strategies. As we un-ravel the biotic and abiotic factors underlying this phenomenon,transkingdom signaling between plants, bacteria, and the preda-tors that feed upon them is expected to play a central role.

ACKNOWLEDGMENTS

We are grateful to D. Haas for plasmid pME6863 and E. Pesci for P.aeruginosa QSC (pEAL01). C. elegans strains were provided by the Caeno-rhabditis Genetics Center, which is funded by the NIH Office of ResearchInfrastructure Programs (grant P40 OD010440).

FUNDING INFORMATIONThis work, including the efforts of Teresa R. de Kievit, was funded byGovernment of Canada | Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) (RGPIN/249559-2012).

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